Because of their unique luminescence properties lanthanide(III) chelates are often used as non-radioactive markers in a wide variety of routine and research applications. For example, U.S. Pat. No. 4,565,670 discloses a heterogeneous method for fluorescence spectroscopic determination of a biologically active substance wherein a lanthanide ion is dissociated from EDTA-labeled immune reactant at low pH-value in solution containing a suitable detergent, a synergistic compound and a β-diketone to amplify the fluorescence after the separation.
Luminescent stable lanthanide(III) chelates has extended the use of time resolution to homogeneous assays. For example, EP 0324 323A discloses an immunochemical assay including a luminescent lanthanide(III) chelate covalently bound to an immune reactant and one or more fluorescence modulating substances such as proteins and detergents.
In the assays described above the detection is based on time-resolved fluorometric analysis of signal derived from a lanthanide(III) chelate including one or more aromatic structures, which absorb the excitation energy and transfer it to the lanthanide(III) ion, and chelating groups such as β-diketones and carboxylic acids. Also tyrosine and phosphotyrosine residues of peptides and proteins are known to sensitize terbium luminescence. These methods relate to the use of a known concentration of peptide residues added to the sample at known concentration. The change in the peptide modification is followed and at the know concentration of enzyme and its activity/binding inhibitors and enhancers. In other words, all concentrations of all molecules in the reaction are known.
US 2011/0111388 discloses a method of detection of an analyte containing polyamino acid and other macromolecules in the aid of luminescent markers that are coupled to the analyte after chemical activation.
U.S. Pat. No. 6,329,205 discloses staining of amine-containing polymers using photoluminescent complexes of europium(III).
The aromatic structures exhibiting the properties described above are also known in art as antennas.
It is known that lanthanide(III) ions can be detected also in the in the absence of antenna molecules. Bekiari et al. have shown that association of lanthanide ions with the ether oxygens of the ethylene glycol groups of PEG chains results in increasing their luminescence intensity in several fluid environments (Chem. Mater. 1999, 11, 3189-3195). The document teaches that such a concentration dependency is achieved for low molecular-weight non-ionic PEG-200 and PEG-400 when measured in the presence of relatively high concentration of lanthanide ion (40 mM) and using time-resolved luminescence detection method. The lowest detectable concentration was approximately 10% (by weight) solution for PEG-200 corresponding to a 0.5 M solution.